EP1125127A2 - Sensor für zielanalyten unter verwendung von mikrosphären - Google Patents

Sensor für zielanalyten unter verwendung von mikrosphären

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Publication number
EP1125127A2
EP1125127A2 EP99945651A EP99945651A EP1125127A2 EP 1125127 A2 EP1125127 A2 EP 1125127A2 EP 99945651 A EP99945651 A EP 99945651A EP 99945651 A EP99945651 A EP 99945651A EP 1125127 A2 EP1125127 A2 EP 1125127A2
Authority
EP
European Patent Office
Prior art keywords
microspheres
sites
substrate
beads
array
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99945651A
Other languages
English (en)
French (fr)
Inventor
David R. Walt
Karri L. Michael
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tufts University
Original Assignee
Tufts University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tufts University filed Critical Tufts University
Publication of EP1125127A2 publication Critical patent/EP1125127A2/de
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54346—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
    • G01N21/7703—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator using reagent-clad optical fibres or optical waveguides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00313—Reactor vessels in a multiple arrangement the reactor vessels being formed by arrays of wells in blocks
    • B01J2219/00315—Microtiter plates
    • B01J2219/00317—Microwell devices, i.e. having large numbers of wells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00646—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports
    • B01J2219/00648—Making arrays on substantially continuous surfaces the compounds being bound to beads immobilised on the solid supports by the use of solid beads
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B01J2219/00659—Two-dimensional arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1434—Optical arrangements
    • G01N2015/1438—Using two lasers in succession
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00584—Control arrangements for automatic analysers
    • G01N2035/0097—Control arrangements for automatic analysers monitoring reactions as a function of time
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S359/00—Optical: systems and elements
    • Y10S359/90—Methods
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/808—Optical sensing apparatus

Definitions

  • optical fibers and optical fiber strands in combination with light absorbing dyes for chemical analytical determinations has undergone rapid development, particularly within the last decade.
  • optical fibers for such purposes and techniques is described by Milanovich et al., "Novel Optical Fiber Techniques For Medical Application", Proceedings of the SPIE 28th Annual International
  • one or more light absorbing dyes are located near its distal end.
  • light from an appropriate source is used to illuminate the dyes through the fiber's proximal end.
  • the light propagates along the length of the optical fiber; and a portion of this propagated light exits the distal end and is absorbed by the dyes.
  • the light absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest; and may or may not be retainable for subsequent use in a second optical determination.
  • fluorophores those more common compositions that emit light after absorption termed "fluorophores” and those which absorb light and internally convert the absorbed light to heat, rather than emit it as light, termed "chromophores.”
  • Fluorescence is a physical phenomenon based upon the ability of some molecules to absorb light (photons) at specified wavelengths and then emit light of a longer wavelength and at a lower energy. Substances able to fluoresce share a number of common characteristics: the ability to absorb light energy at one wavelength ⁇ ab ; reach an excited energy state; and subsequently emit light at another light wavelength, ⁇ em .
  • the absorption and fluorescence emission spectra are individual for each fluorophore and are often graphically represented as two separate curves that are slightly overlapping.
  • the same fluorescence emission spectrum is generally observed irrespective of the wavelength of the exciting light and, accordingly, the wavelength and energy of the exciting light may be varied within limits; but the light emitted by the fluorophore will always provide the same emission spectrum.
  • the strength of the fluorescence signal may be measured as the quantum yield of light emitted.
  • the fluorescence quantum yield is the ratio of the number of photons emitted in comparison to the number of photons initially absorbed by the fluorophore.
  • Dyes which absorb light energy as chromophores do so at individual wavelengths of energy and are characterized by a distinctive molar absorption coefficient at that wavelength.
  • Chemical analysis employing fiber optic strands and absorption spectroscopy using visible and ultraviolet light wavelengths in combination with the absorption coefficient allow for the determination of concentration for specific analyses of interest by spectral measurement.
  • absorbance measurement via optical fibers is to determine concentration which is calculated in accordance with Beers' law; accordingly, at a single absorbance wavelength, the greater the quantity of the composition which absorbs light energy at a given wavelength, the greater the optical density for the sample In this way, the total quantity of light absorbed directly correlates with the quantity of the composition in the sample
  • compositions and methods are desirable that allow the generation of large fiber optic arrays including microspheres that can be either encoded or decoded to allow the detection of target analytes
  • compositions comprising a substrate with a surface comprising discrete sites, and a population of microspheres distributed on the sites
  • the sites may be wells or chemically functionalized sites
  • the invention provides methods of determining the presence of a target analyte in a sample
  • the methods comprise contacting the sample with a composition comprising a substrate with a surface comprising discrete sites, and a population of microspheres comprising at least a first and a second subpopulation Each subpopulation comprises a bioactive agent and an optical signature capable of identifying the bioactive agent
  • the microspheres are distributed on the surface such that the discrete sites contain microspheres The presence or absence of the target analyte is then determined
  • the invention provides methods of making a composition
  • a composition comprising forming a surface comprising individual sites on a substrate, distributing microspheres on the surface such that the individual sites contain microspheres
  • the microspheres comprise at least a first and a second subpopulation, each comprising a bioactive agent, and an optical signature capable of identifying said bioactive agent
  • Fig 1 is a schematic diagram illustrating the optical signature encoding and chemical functionalizing of the microspheres according to the present invention
  • Fig 2 is a process diagram describing the preparation, encoding, and functionalizing of the microspheres of the present invention
  • Fig 3 is a schematic diagram illustrating a microsphere system including microspheres with different chemical functionalities and encoded descriptions of the functionalities
  • Fig 4 is a schematic diagram of the inventive fiber optic sensor and associated instrumentation and control system
  • Fig 5A and 5B are micrographs illustrating the preferred technique for attaching or affixing the microspheres to the distal end of the optical fiber bundle
  • Fig 6 is a process diagram describing well formation in the optical fiber bundle and affixation of the microspheres in the wells
  • Figs 7A and 7B are micrographs showing the array of microspheres in their corresponding wells prior and subsequent to physical agitation, tapping and air pulsing, demonstrating the electrostatic binding of the microspheres in the wells,
  • Figs 8A, 8B, and 8C are micrographs from alkaline phosphatase microspheres when exposed to fluorescein diphosphate, at the fluorescein emission wavelength, at an encoding wavelength for DilC, and at an encoding wavelength for TRC, respectively,
  • Figs 9A and 9B are micrographs showing the optical signal from ⁇ -galactosidase microspheres when exposed to fluorescein ⁇ -galactopyranoside at the fluorescein emission wavelength and at an encoding wavelength for DilC, respectively, and
  • Fig 10A and 10B are micrographs showing the optical response from rabbit antibody microspheres prior to and post, respectively, exposure to fluorescein labeled antigens
  • Fig 11 A and 11 B are micrographs depicting the optical response from beads synthesized with DNA on the bead surface, following a 10 mm hybridization with a Cy3-labeled probe complementary to the sequence of the DNA immobilized on the bead Beads were randomly distributed on A) an etched optical imaging fiber or B) a patterned polymer (polyurethane) substrate (a chip) Following hybridization with 5 nM Cy3-labeled probe, the substrates were placed in buffer for optical readout on an imaging system A) was imaged through the proximal end, with the distal (beaded) end in buffer solution B) was imaged directly from the top, through a covers p
  • Fig 12A, 12B and 12C are micrographs depicting the optical responses between different substrates
  • the substrate in A) and B) is an etched optical imaging fiber, and the substrate in C) is a chip Data were obtained as described in Fig 11 , and quantified to determine mean intensity and variability
  • the present invention is based on two synergistic inventions 1) the development of a bead-based analytic chemistry system in which beads, also termed microspheres, carrying different chemical functionalities may be mixed together while the ability is retained to identify the functionality of each bead using an optically interrogatable encoding scheme (an "optical signature"), and 2) the use of a substrate comprising a patterned surface containing individual sites that can bind or associate individual beads
  • an optical signature optically interrogatable encoding scheme
  • the present invention provides array compositions comprising at least a first substrate with a surface comprising individual sites
  • array herein is meant a plurality of bioactive agents in an array format, the size of the array will depend on the composition and end use of the array Arrays containing from about 2 different bioactive agents (i e different beads) to many millions can be made, with very large fiber optic arrays being possible
  • the array will comprise from two to as many as a billion or more, depending on the size of the beads and the substrate, as well as the end use of the array, thus very high density, high density, moderate density, low density and very low density arrays may be made
  • Preferred ranges for very high density arrays are from about 10,000,000 to about 2,000,000,000, with from about 100,000,000 to about 1 ,000,000,000 being preferred
  • High density arrays range about 100,000 to about 10,000,000, with from about 1,000,000 to about 5,000,000 being particularly preferred
  • Moderate density arrays range from about 10,000 to about 50,000 being particularly preferred, and from about 20,000 to about 30,000 being especially preferred
  • Low density arrays are generally less than 10,000, with from about 1 ,000 to about 5,000 being preferred Very low density arrays are less than 1,000, with from about 10 to about 1000 being preferred, and from about 100 to about 500 being particularly preferred
  • the compositions of the invention may not be in array format, that is, for some embodiments, compositions comprising a single bioactive agent may be made as well
  • multiple substrates may be used, either of different or identical compositions
  • large arrays may comprise a plurality of smaller substrates
  • one advantage of the present compositions is that particularly through the use of fiber optic technology, extremely high density arrays can be made.
  • beads of 200 nm can be used, and very small fibers are known, it is possible to have as many as 250,000 different fibers and beads in a 1 mm 2 fiber optic bundle, with densities of greater than 15,000,000 individual beads and fibers per 0 5 cm 2 obtainable
  • compositions comprise a substrate
  • substrate or “solid support” or other grammatical equivalents herein is meant any material that can be modified to contain discrete individual sites appropriate for the attachment or association of beads and is amenable to at least one detection method
  • substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc ), polysaccha ⁇ des, nylon or nitrocellulose, resins, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, optical fiber bundles, and a variety of other polymers
  • the substrates allow optical detection and do not appreciably fluorescese
  • the substrate does not comprise an optical fiber bundle or array
  • the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well, for example, three dimensional configurations can be used, for example by embedding the beads in a porous block of plastic that allows sample access to the beads and using a confocal microscope for detection Similarly, the beads may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume
  • Preferred substrates include optical fiber bundles as discussed below, and flat planar substrates such as glass, polystyrene and other plastics and acrylics
  • At least one surface of the substrate is modified to contain discrete, individual sites for later association of microspheres
  • These sites may comprise physically altered sites, i e physical configurations such as wells or small depressions in the substrate that can retain the beads, such that a microsphere can rest in the well, or the use of other forces (magnetic or compressive), or chemically altered or active sites, such as chemically functionalized sites, electrostatically altered sites, hydrophobically/ hydrophilically functionalized sites, spots of adhesive, etc
  • the sites may be a pattern, i e a regular design or configuration, or randomly distributed
  • a preferred embodiment utilizes a regular pattern of sites such that the sites may be addressed in the X-Y coordinate plane "Pattern" in this sense includes a repeating unit cell, preferably one that allows a high density of beads on the substrate
  • these sites may not be discrete sites That is, it is possible to use a uniform surface of adhesive or chemical functionalities, for example, that allows the attachment of beads at any position That is, the surface of the substrate is modified to allow attachment of the microspheres at individual sites, whether or not those sites are contiguous or non-contiguous with other sites
  • the surface of the substrate may be modified such that discrete sites are formed that can only have a single associated bead, or alternatively, the surface of the substrate is modified and beads may go down anywhere, but they end up at discrete sites
  • the surface of the substrate is modified to contain wells, i e depressions in the surface of the substrate. This may be done as is generally known in the art using a variety of techniques, including, but not limited to, photolithography, stamping techniques, molding techniques and microetching techniques As will be appreciated by those in the art, the technique used will depend on the composition and shape of the substrate
  • the substrate is a fiber optic bundle and the surface of the substrate is a terminal end of the fiber bundle
  • wells are made in a terminal or distal end of a fiber optic bundle comprising individual fibers
  • the cores of the individual fibers are etched, with respect to the cladding, such that small wells or depressions are formed at one end of the fibers The required depth of the wells will depend on the size of the beads to be added to the wells
  • the microspheres are non-covalently associated in the wells, although the wells may additionally be chemically functionalized as is generally described below, cross-linking agents may be used, or a physical barrier may be used, i e a film or membrane over the beads
  • the surface of the substrate is modified to contain chemically modified sites, that can be used to attach, either covalently or non-covalently, the microspheres of the invention to the discrete sites or locations on the substrate "Chemically modified sites" in this context includes, but is not limited to, the addition of a pattern of chemical functional groups including ammo groups, carboxy groups, oxo groups and thiol groups, that can be used to covalently attach microspheres, which generally also contain corresponding reactive functional groups, the addition of a pattern of adhesive that can be used to bind the microspheres (either by prior chemical functionalization for the addition of the adhesive or direct addition of the adhesive), the addition of a pattern of charged groups (similar to the chemical functionalities) for the electrostatic attachment of the microspheres, i e when the microspheres comprise charged groups opposite to the sites, the addition of a pattern of chemical functional groups that renders the sites differentially hydrophobic or hydrophilic, such that the addition of similarly hydrophobic or hydrophilic microspheres under
  • compositions of the invention further comprise a population of microspheres
  • population herein is meant a plurality of beads as outlined above for arrays Within the population are separate subpopulations, which can be a single microsphere or multiple identical microspheres That is, in some embodiments, as is more fully outlined below, the array may contain only a single bead for each bioactive agent, preferred embodiments utilize a plurality of beads of each type
  • microspheres or “beads” or “particles” or grammatical equivalents herein is meant small discrete particles
  • the composition of the beads will vary, depending on the class of bioactive agent and the method of synthesis Suitable bead compositions include those used in peptide, nucleic acid and organic moiety synthesis, including, but not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, tho ⁇ a sol, carbon graphited, titanium dioxide, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, cross-linked micelles and teflon may all be used "Microsphere Detection Guide” from Bangs Laboratories, Fishers IN is a helpful guide
  • the beads need not be spherical, irregular particles may be used
  • the beads may be porous, thus increasing the surface area of the bead available for either bioactive agent attachment or tag attachment
  • the bead sizes range from nanometers, i e 100 nm, to millimeters, i e 1 mm, with beads from about 0 2 micron to about 200 microns being preferred, and from about 0 5 to about 5 micron being particularly preferred, although in some embodiments smaller beads may be used
  • Fig 1 illustrates the construction of a bead or microsphere 10 according to the principles of the present invention
  • the microsphere 10 is given a bioactive agent 12, which is typically applied to the microsphere's surface
  • the bioactive agent is designed so that in the presence of the analyte(s) to which it is targeted, an optical signature of the microsphere, possibly including region surrounding it, is changed
  • a key component of the invention is the use of a substrate/bead pairing that allows the association or attachment of the beads at discrete sites on the surface of the substrate, such that the beads do not move during the course of the assay
  • Each microsphere comprises two components a bioactive agent and an optical signature
  • compositions of the invention have two primary uses
  • the compositions are used to detect the presence of a particular target analyte, for example, the presence or absence of a particular nucleotide sequence or a particular protein, such as an enzyme, an antibody or an antigen
  • the compositions are used to screen bioactive agents, i e drug candidates, for binding to a particular target analyte
  • Bioactive agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons
  • Bioactive agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups
  • the bioactive agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups
  • Bioactive agents are also found among biomolecules including peptides, nucleic acids, sacchandes, fatty acids, steroids, punnes, pynmidines, derivatives, structural analogs or combinations thereof Particularly preferred are nucleic acids and proteins
  • Bioactive agents can be obtained from a wide variety of sources including libraries of synthetic or natural compounds For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, este ⁇ fication and/or amidification to produce structural analogs
  • the bioactive agents are proteins
  • protein herein is meant at least two covalently attached ammo acids, which includes proteins, polypeptides, o gopeptides and peptides
  • the protein may be made up of naturally occurring ammo acids and peptide bonds, or synthetic peptidomimetic structures
  • “ammo acid”, or “peptide residue”, as used herein means both naturally occurring and synthetic ammo acids
  • homo-phenylalanine, citrulline and norleucme are considered ammo acids for the purposes of the invention
  • the side chains may be in either the (R) or the (S) configuration
  • the ammo acids are in the (S) or L-configuration If non-naturally occurring side chains are used, non-ammo acid substituents may be used, for example to prevent or retard in vivo degradations
  • the bioactive agents are naturally occurring proteins or fragments of naturally occunng proteins
  • cellular extracts containing proteins, or random or directed digests of protemaceous cellular extracts may be used
  • libraries of procaryotic and eukaryotic proteins may be made for screening in the systems described herein
  • Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred
  • the bioactive agents are peptides of from about 5 to about 30 ammo acids, with from about 5 to about 20 ammo acids being preferred, and from about 7 to about 15 being particularly preferred
  • the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides
  • randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and ammo acids, respectively Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or ammo acid at any position
  • the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized bioactive protemaceous agents
  • a library of bioactive agents are used The library should provide a sufficiently structurally diverse population of bioactive agents to effect a probabilistically sufficient range of binding to target analytes Accordingly, an interaction library must be large enough so that at least one of its members will have a structure that gives it affinity for the target analyte Although it is difficult to gauge the required absolute size of an interaction library, nature provides a hint with the immune response a diversity of 10 7 -10 8 different antibodies provides at least one combination with sufficient affinity to interact with most potential antigens faced by an organism Published in vitro selection techniques have also shown that a library size of 10 7 to 10 ⁇ is sufficient to find structures with affinity for the target Thus, in a preferred embodiment, at least 10 6 , preferably at least 10 7 , more preferably at least 10 8 and most preferably at least 10 9 different bioactive agents are simultaneously analyzed in the subject methods Preferred methods maximize library size and diversity In one embodiment, the library is fully randomized, with no sequence preferences or constants at any position.
  • the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
  • the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, pralines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
  • the bioactive agents are nucleic acids (generally called “probe nucleic acids” or “candidate probes” herein).
  • nucleic acid or “oligonucleotide” or grammatical equivalents herein means at least two nucleotides covalently linked together.
  • a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage, et al., Tetrahedron, 49(10):1925 (1993) and references therein; Letsinger, J. Or ⁇ . Chem..
  • nucleic acids include those with positive backbones (Denpcy, et al., Proc Natl. Acad. Sci. USA. 92:6097 (1995)); non-ionic backbones (U.S. Patent Nos. 5,386,023; 5,637,684; 5,602,240; 5,216,141; and 4,469,863; Kiedrowshi, et al., Angew. Chem. Intl. Ed. English. 30:423 ( 1991 ) ; Letsinger, ef al. , J. Am. Chem. Soc. 110:4470 ( 1988) ;
  • nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins, et al., Chem. Soc. Rev.. (1995) pp. 169-176).
  • nucleic acid analogs are described in Rawls, C & E News, June 2, 1997, page 35. All of these references are hereby expressly incorporated by reference.
  • nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence
  • the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxy ⁇ bo- and ⁇ bo-nucleotides, and any combination of bases, including uracil, adenme, thymme, cytosme, guanme, inosme, xanthan e, hypoxanthanine, isocytosme, isoguanme, and basepair analogs such as
  • nucleic acid bioactive agents may be naturally occunng nucleic acids, random nucleic acids, or "biased" random nucleic acids
  • digests of procaryotic or eukaryotic genomes may be used as is outlined above for proteins
  • probes of the present invention are designed to be complementary to a target sequence (either the target analyte sequence of the sample or to other probe sequences, as is described herein), such that hybridization of the target and the probes of the present invention occurs
  • a target sequence either the target analyte sequence of the sample or to other probe sequences, as is described herein
  • This complementarity need not be perfect, there may be any number of base pair mismatches that will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention
  • the sequence is not a complementary target sequence
  • substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under the selected reaction conditions
  • High stringency conditions are known in the art, see for example Maniatis et al , Molecular Cloning A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed Ausubel, et al , both of which are hereby incorporated by reference
  • Stringent conditions are
  • target sequence' or grammatical equivalents herein means a nucleic acid sequence on a single strand of nucleic acid
  • the target sequence may be a portion of a gene, a regulatory sequence, genomic DNA, cDNA, RNA including mRNA and rRNA, or others It may be any length, with the understanding that longer sequences are more specific
  • the complementary target sequence may take many forms For example, it may be contained within a larger nucleic acid sequence, i e all or part of a gene or mRNA, a restriction fragment of a plasmid or genomic DNA, among others
  • probes are made to hybridize to target sequences to determine the presence or absence of the target sequence in a sample Generally speaking, this term will be understood by those skilled in the art
  • the bioactive agents are organic chemical moieties, a wide variety of which are available in the literature
  • each bead comprises a single type of bioactive agent, although a plurality of individual bioactive agents are preferably attached to each bead
  • preferred embodiments utilize more than one microsphere containing a unique bioactive agent, that is, there is redundancy built into the system by the use of subpopulations of microspheres, each microsphere in the subpopulation containing the same bioactive agent
  • the bioactive agents may either be synthesized directly on the beads, or they may be made and then attached after synthesis
  • linkers are used to attach the bioactive agents to the beads, to allow both good attachment, sufficient flexibility to allow good interaction with the target molecule, and to avoid undesirable binding reactions
  • the bioactive agents are synthesized directly on the beads
  • many classes of chemical compounds are currently synthesized on solid supports, such as peptides, organic moieties, and nucleic acids It is a relatively straightforward matter to adjust the current synthetic techniques to use beads
  • the bioactive agents are synthesized first, and then covalently attached to the beads.
  • this will be done depending on the composition of the bioactive agents and the beads
  • the functionalization of solid support surfaces such as certain polymers with chemically reactive groups such as thiols, amines, carboxyls, etc is generally known in the art Accordingly, "blank" microspheres may be used that have surface chemistries that facilitate the attachment of the desired functionality by the user Some examples of these surface chemistries for blank microspheres are listed in Table I
  • bioactive agents containing carbohydrates may be attached to an ammo-functionalized support, the aldehyde of the carbohydrate is made using standard techniques, and then the aldehyde is reacted with an ammo group on the surface
  • a sulfhydryl linker may be used
  • sulfhydryl reactive linkers such as SPDP, maleimides, ⁇ -haloacetyls, and pyndyl disulfides (see for example the 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference) which can be used to attach cysteme containing protemaceous agents to the support
  • an ammo group on the bioactive agent may be used for attachment to an ammo group on the surface
  • a large number of stable bifunctional groups are well known in the art, including homo
  • bioactive agents may be attached in a variety of ways, including those listed above What is important is that manner of attachment does not significantly alter the functionality of the bioactive agent, that is, the bioactive agent should be attached in such a flexible manner as to allow its interaction with a target
  • NH 2 surface chemistry microspheres are used Surface activation is achieved with a 2 5% glutaraldehyde in phosphate buffered saline (10 mM) providing a pH of 6 9 (138 mM NaCI, 2 7 mM,
  • the microspheres comprise an optical signature that can be used to identify the attached bioactive agent That is, each subpopulation of microspheres comprise a unique optical signature or optical tag that can be used to identify the unique bioactive agent of that subpopulation of microspheres, a bead comprising the unique optical signature may be distinguished from beads at other locations with different optical signatures As is outlined herein, each bioactive agent will have an associated unique optical signature such that any microspheres comprising that bioactive agent will be identifiable on the basis of the signature As is more fully outlined below, it is possible to reuse or duplicate optical signatures within an array, for example, when another level of identification is used, for example when beads of different sizes are used, or when the array is loaded sequentially with different batches of beads
  • the optical signature is generally a mixture of reporter dyes, preferably fluoroscent
  • matrices of unique tags may be generated This may be done by covalently attaching the dyes to the surface of the beads, or alternatively, by entrapping the dye within the bead
  • the dyes may be chromophores or phosphors but are preferably fluorescent dyes, which due to their strong signals provide a good signal-to-noise ratio for decoding
  • Suitable dyes for use in the invention include, but are not limited to, fluorescent lanthanide complexes, including those of Europium and Terbium, fluorescein, rhodamme, tetramethylrhodamine, eosm, erythrosin, coumarm, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cas
  • the encoding can be accomplished in a ratio of at least two dyes, although more encoding dimensions may be added in the size of the beads, for example
  • the labels are distinguishable from one another, thus two different labels may comprise different molecules (i e two different fluors) or, alternatively, one label at two different concentrations or intensity
  • the dyes are covalently attached to the surface of the beads This may be done as is generally outlined for the attachment of the bioactive agents, using functional groups on the surface of the beads As will be appreciated by those in the art, these attachments are done to minimize the effect on the dye
  • the dyes are non-covalently associated with the beads, generally by entrapping the dyes in the bead matrix or pores of the beads
  • reporter dyes 14 are added to the microsphere 10 with the encoding occurring in the ratio of two or more dyes
  • the reporter dyes 14 may be chromophore-type Fluorescent dyes, however, are preferred because the strength of the fluorescent signal provides a better signal-to-noise ratio when decoding Additionally, encoding in the ratios of the two or more dyes, rather than single dye concentrations, is preferred since it provides msensitivity to the intensity of light used to interrogate the reporter dye's signature and detector sensitivity
  • the dyes are added to the bioactive agent, rather than the beads, although this is generally not preferred
  • Fig 2 is a process diagram illustrating the preparation of the microspheres
  • step 50 an aliquot of stock microspheres are vacuum filtered to produce a dry cake
  • microsphere copolymers of methylstyrene (87%) and divmylbenzene (13%) are used that have a 3 1 micrometer ( ⁇ m) diameter
  • Dyes may be covalently bonded to the microspheres' surface, but this consumes surface binding sites desirably reserved for the chemical functionalities
  • the microspheres are placed in a dye solution comprising a ratio of two or more fluorescent reporter dyes dissolved in an organic solvent that will swell the microspheres, e g , dimethylformamide (DMF)
  • DMF dimethylformamide
  • Examples of other dyes that can be used are Oxazin (662/705), IR-144 (745/825), IR-140 (776/882), IR-125 (786/800) from Exciton, and Bodipy 665/676 from Molecular Probes, and Naphthofluorescem (605/675) also from Molecular Probes Lathanide complexes may also be used Fluorescent dyes emitting in other than the near infrared may also be used Chromophore dyes are still another alternative that produce an optically interrogatable signature, as are more exotic formulations using Raman scattering-based dyes or polarizing dyes, for example The ability of a particular dye pair to encode for different chemical functionalities depends on the resolution of the ratiometnc measurement Conservatively, any dye pair should provide the ability to discriminate at least twenty different ratios The number of unique combinations of two dyes made with a particular dye set is shown in the following Table II
  • step 54 the microspheres are vacuum filtered to remove excess dye The microspheres are then washed in water or other liquid that does not swell the microspheres, but in which the dyes are still soluble This allows the residual dye to be rinsed off without rinsing the dye out of the microspheres
  • step 56 the bioactive agent is attached to the microsphere surface if not already present It should be understood that surface chemistries may be present throughout the microsphere's volume, and not limited to the physical circumferential surface
  • the microspheres are added to discrete sites on the surface of the substrate
  • the association of the beads on the surface may comprise a covalent bonding of the bead to the surface, for example when chemical attachment sites are added to both the substrate and the bead, an electrostatic or hydroaffinity, when charge, hydrophobicity or hydrophilicity is used as the basis of the binding, a physical yet non-covalent attachment such as the use of an adhesive, or a spatial attachment, for example the localization of a bead within a well
  • it may be preferable to effect a more permanent attachment after the initial localization for example through the use of cross-linking agents, a film or membrane over the array
  • Fig 3 schematically illustrates a microsphere system, or array of microspheres, 100 formed from microsphere populations that have different bioactive agents While a large number of microspheres and bioactive agents may be employed, in this example only three microsphere populations are shown
  • the individual populations, or subpopulations, of microspheres are represented as IOa,IOb,IOc carrying respective bioactive agents or probe sequences 60a,60b,60c, as exemplary functionalities
  • the subpopulations may be combined in either a random or ordered fashion on a substrate, with a corresponding distribution of their respective bioactive agents
  • each microsphere in each subpopulation is encoded with a common optical signature
  • the subpopulation represented by microsphere 10a has a two reporter dye ratio of 10 1
  • the subpopulation of microspheres 10b has a ratio of 1 1 of the same reporter dyes
  • subpopulation of microspheres 10c has a ratio of 1 10 of the reporter dyes
  • the randomly mixed subpopulations of microspheres are useful as an analytic chemistry system based on each of the carried bioactive agents 60a-60c separately
  • the microsphere array or system 100 is exposed to an analyte of interest to which some of the bioactive agents may interact Any interaction changes the optical response of the corresponding microspheres by, for example, binding a fluorescent dye 64 to the microspheres
  • identifying the chemical functionalities of the microsphere in which the optical signature has changed using the encoded dye combinations, information regarding the chemical identity and concentration of an analyte may be gained based upon the interaction or noninteraction of each bioactive agent contained in the microsphere system 100
  • microspheres exhibiting activity or changes in their optical signature may be identified by a conventional optical tram and optical detection system Decoding can also be performed either manually or automatically with the aid of a computer Depending on the particular encoding or reporter dyes used and their operative wavelengths, optical filters designed for a particular wavelengths may be employed for optical interrogation of the microspheres of bioactive agents In a preferred embodiment, the analytic chemistry microsphere system is used in conjunction with an optical fiber bundle or fiber optic array as a substrate
  • Fig 4 is a schematic block diagram showing a microsphere-based analytic chemistry system employing a fiber optic assembly 200 with an optical detection system
  • the fiber optic assembly 200 comprises a fiber optic bundle or array 202, that is constructed from clad fibers so that light does not mix between fibers
  • a microsphere array or system, 100 is attached to the bundle's distal end 212, with the proximal end 214 being received by a z-translation stage 216 and x-y m ⁇ cropos ⁇ t ⁇ oner 218
  • These two components act in concert to properly position the proximal end 214 of the bundle 202 for a microscope objective lens 220
  • Light collected by the objective lens 220 is passed to a reflected light fluorescence attachment with three pointer cube slider 222
  • the attachment 222 allows insertion of light from a 75 Watt Xe lamp 224 through the objective lens 220 to be coupled into the fiber bundle 202
  • the light from the source 224 is condensed by condensing lens 226,
  • the microsphere array or system 100 may be attached to the distal end of the optical fiber bundle using a variety of compatible processes It is important that the microspheres are located close to the end of the bundle This ensures that the light returning in each optical fiber predominantly comes from only a single microsphere This feature is necessary to enable the interrogation of the optical signature of individual microspheres to identify reactions involving the microsphere's functionality and also to decode the dye ratios contained in those microspheres
  • the adhesion or affixing technique must not chemically insulate the microspheres from the analyte
  • Figs 5A and 5B are micrographs of the distal end 212 of the bundle 202 illustrating the preferred technique for attaching the microspheres 10 to the bundle 202.
  • Wells 250 are formed at the center of each optical fiber 252 of the bundle 202.
  • the size of the wells 250 are coordinated with the size of the microspheres 10 so that the microspheres 10 can be placed within the wells 250.
  • each optical fiber 252 of the bundle 202 conveys light from the single microsphere 10 contained in 5 its well Consequently, by imaging the end of the bundle 202 onto the CCD array 236, the optical signatures of the microspheres 10 are individually interrogatable
  • Fig 6 illustrates how the microwells 250 are formed and microspheres 10 placed in the wells
  • a 1 mm hexagonally-packed imaging fiber contains approximately 20,600 individual optical fibers that have cores approximately 3 7 ⁇ m across (Part No ET26 from Galileo Fibers)
  • the cores of each fiber are hexagonally shaped as a result the starting preform, that is, during drawing the fiber does not usually change shape In some cases, the shape can be circular, however
  • both the proximal and distal ends 212,214 of the fiber bundle 202 are successively polished on 12 ⁇ m, 9 ⁇ m, 3 ⁇ m, 1 ⁇ m, and 0 3 ⁇ m lapping films Subsequently, the ends can be inspected for scratches on an atomic force microscope
  • a representative etching is performed on the distal end 212 of the bundle 202.
  • a solution of 0 2 grams NH 4 F (ammonium fluoride) with 600 ⁇ l distilled H 2 0 and 100 ⁇ 1 of HF (hydrofluoric acid), 50% stock solution, may be used The distal end 212 is etched in this solution for a specified time, preferably approximately 30 to 600 seconds, with about 80 seconds being preferred
  • the bundle end Upon removal from this solution, the bundle end is immediately placed in deionized water to stop any further etching in step 274.
  • the fiber is then rinsed in running tap water At this stage, sonication is preferably performed for several minutes to remove any salt products from the reaction The fiber is then allowed to air dry
  • the foregoing procedure produces wells by the anisotr opic etching of the fiber cores 254 favorably with respect to the cladding 256 for each fiber of the bundle 202.
  • the wells have approximately the diameter of the cores 254, 3 7 ⁇ m This diameter is selected to be slightly larger than the diameters of the microspheres used, 3 1 ⁇ m, in the example
  • the preferential etching occurs because the pure silica of the cores 254 etches faster in the presence of hydrofluoric acid than the germanium-doped silica claddings 256.
  • microspheres are then placed in the wells 250 in step 276 according to a number of different techniques
  • the placement of the microspheres may be accomplished by dripping a solution containing the desired randomly mixed subpopulations of the microspheres over the distal end 212, sonicating the bundle to settle the microspheres in the wells, and allowing the microsphere solvent to evaporate
  • the subpopulations could be added serially to the bundle end
  • Microspheres 10 may then be fixed into the wells 250 by using a dilute solution of sulfonated Nation that is dripped over the end Upon solvent evaporation, a thin film of Nafion was formed over the microspheres which holds them in place
  • This approach is compatible for fixing microspheres for pH indication that carry FITC functionality
  • the resulting array of fixed microspheres retains its pH sensitivity due to the permeability of the sulfonated Nafion to hydrogen ions This approach, however, can not be employed genencally as Nafion is imper
  • microsphere swelling to entrap each microsphere 10 in its corresponding microwell 250
  • the microspheres are first distributed into the microwells 250 by sonicating the microspheres suspended in a non-swelling solvent in the presence of the microwell array on the distal end 212. After placement into the microwells, the microspheres are subsequently exposed to an aqueous buffer in which they swell, thereby physically entrapping them, analogous to muffins rising in a muffin tin
  • compositions of the invention may be made in a variety of ways
  • the arrays are made by adding a solution or slurry comprising the beads to a surface containing the sites for attachment of the beads This may be done in a variety of buffers, including aqueous and organic solvents, and mixtures The solvent can evaporate, and excess beads removed
  • a novel method of loading the beads onto the array comprises exposing the array to a solution of particles (including microspheres and cells) and then applying energy, e g agitating or vibrating the mixture This results in an array comprising more tightly associated particles, as the agitation is done with sufficient energy to cause weakly-associated beads to fall off (or out, in the case of wells) These sites are then available to bind a different bead In this way, beads that exhibit a high affinity for the sites are selected Arrays made in this way have two mam advantages as compared to a more static loading first of all, a higher percentage of the sites can be filled easily, and secondly, the arrays thus loaded show a substantial decrease in bead loss during assays
  • these methods are used to generate arrays that have at least about 50% of the sites filled, with at least about 75% being preferred, and at least about 90% being particularly preferred Similarly, arrays
  • the substrate comprising the surface with the discrete sites is immersed into a solution comprising the particles (beads, cells, etc )
  • the surface may comprise wells, as is described herein, or other types of sites on a patterned surface such that there is a differential affinity for the sites This diffemetial affinity results in a competitive process, such that particles that will associate more tightly are selected
  • the entire surface to be "loaded” with beads is in fluid contact with the solution
  • This solution is generally a slurry ranging from about 10,000 1 beads solution (vol vol) to 1 1 1
  • the solution can comprise any number of reagents, including aqueous buffers, organic solvents, salts, other reagent components, etc
  • the solution preferably comprises an excess of beads, that is, there are more beads than sites on the array Preferred embodiments utilize two-fold to billion-fold excess of beads
  • the immersion can mimic the assay conditions, for example, if the array is to be "dipped" from above into a microtiter plate comprising samples, this configuration can be repeated for the loading, thus minimizing the beads that are likely to fall out due to gravity
  • the substrate, the solution, or both are subjected to a competitive process, whereby the particles with lower affinity can be disassociated from the substrate and replaced by particles exhibiting a higher affinity to the site
  • This competitive process is done by the introduction of energy, in the form of heat, sonication, stirring or mixing, vibrating or agitating the solution or substrate, or both
  • a preferred embodiment utilizes agitation or vibration
  • the amount of manipulation of the substrate is minimized to prevent damage to the array
  • preferred embodiments utilize the agitation of the solution rather than the array, although either will work
  • this agitation can take on any number of forms, with a preferred embodiment utilizing microtiter plates comprising bead solutions being agitated using microtiter plate shakers
  • the agitation proceeds for a period of time sufficient to load the array to a desired fill Depending on the size and concentration of the beads and the size of the array, this time may range from about 1 second to days, with from about 1 minute to about 24 hours being preferred
  • sites of an array may comprise a bead, that is, there may be some sites on the substrate surface which are empty.
  • microspheres One of the most common microsphere formations is tentagel, a styrene-polyethylene glycol co-polymer These microspheres are unswollen in nonpolar solvents such as hexane and swell approximately 20-40% in volume upon exposure to a more polar or aqueous media This approach is extremely desirable since it does not significantly compromise the diffusional or permeability properties of the microspheres themselves
  • Figs 7A and 7B show polymer coated microspheres 12 in wells 250 after their initial placement and then after tapping and exposure to air pulses
  • Figs 7A and 7B illustrate that there is no appreciable loss of microspheres from the wells due to mechanical agitation even without a specific fixing technique This effect is probably due to electrostatic forces between the microspheres and the optical fibers These forces tend to bind the microspheres within the wells Thus, in most environments, it may be unnecessary to use any chemical or mechanical fixation for the microspheres
  • a sonication step may be used to place beads in the wells
  • sites of an array may comprise a bead, that is, there may be some sites on the substrate surface which are empty In addition, there may be some sites that contain more than one bead, although this is not preferred In some embodiments, for example when chemical attachment is done, it is possible to attach the beads in a non-random or ordered way For example, using photoactivatible attachment linkers or photoactivatible adhesives or masks, selected sites on the array may be sequentially rendered suitable for attachment, such that defined populations of beads are laid down
  • the size of the array will be set by the number of unique optical signatures, it is possible to "reuse" a set of unique optical signatures to allow for a greater number of test sites This may be done in several ways, for example, by using a positional coding scheme within an array, different sub-bundles may reuse the set of optical signatures Similarly, one embodiment utilizes bead size as a coding modality, thus allowing the reuse of the set of unique optical signatures for each bead size Alternatively, sequential partial loading of arrays with beads can also allow the reuse of optical signatures
  • a spatial or positional coding system is done
  • each subarray is an "area code", that can have the same tags (i e telephone numbers) of other subarrays, that are separated by virtue of the location of the subarray
  • tags i e telephone numbers
  • the same unique tags can be reused from bundle to bundle
  • the use of 50 unique tags in combination with 100 different subarrays can form an array of 5000 different bioactive agents
  • additional encoding parameters can be added, such as microsphere size
  • the use of different size beads may also allow the reuse of sets of optical signatures, that is, it is possible to use microspheres of different sizes to expand the encoding dimensions of the microspheres
  • Optical fiber arrays can be fabricated containing pixels with different fiber diameters or cross-sections, alternatively, two or more fiber optic bundles, each with different cross-sections of the individual fibers, can be added together to form a larger bundle, or, fiber optic bundles with fiber of the same size cross-sections can be used, but just with different sized beads With different diameters, the largest wells can be filled with the largest microspheres and then moving onto progressively smaller microspheres in the smaller wells until all size wells are then filled In this manner, the same dye ratio could be used to encode microspheres of different sizes thereby expanding the number of different oligonucleotide sequences or chemical functionalities present in the array
  • fiber optic substrates this as well as the other methods outlined
  • arrays are made of a large spectrum of chemical functionalities utilizing the compositions of invention comprising microspheres and substrates with discrete sites on a surface
  • prior art sensors which can be adapted for use in the present invention include four broad classifications of microsphere sensors 1) basic indicator chemistry sensors, 2) enzyme-based sensors, 3) immuno-based sensors (both of which are part of a broader general class of protein sensors), and 4) geno-sensors
  • bioactive agents are used to detect chemical compounds
  • a large number of basic indicator sensors have been previously demonstrated Examples include
  • Enzyme-based microsphere sensors have also been demonstrated and could be manifest on microspheres Examples include
  • the induced change in the optical signal due to the presence of the enzyme-sensitive chemical analyte occurs indirectly in this class of chemical functionalities
  • the microsphere-bound enzyme e g , glucose oxidase
  • decomposes the target analyte, e g , glucose consume a co-substrate, e g , oxygen, or produce some by-product, e g , hydrogen peroxide
  • An oxygen sensitive dye is then used to trigger the signal change
  • Immuno-based microsphere sensors have been demonstrated for the detection for environmental pollutants such as pesticides, herbicides, PCB's and PAH's Additionally, these sensors have also been used for diagnostics, such as bacterial (e g , leprosy, cholera, lyme disease, and tuberculosis), viral (e , HIV, herpes simplex, cytomegalovirus), fungal (e g , aspergillosis, candidiasis, cryptococcoses
  • Microsphere genosensors may also be made (see the Examples) These are typically constructed by attaching a probe sequence to the microsphere surface chemistry, typically via an NH 2 group A fluorescent dye molecule, e , fluorescein, is attached to the target sequence, which is in solution The optically interrogatable signal change occurs with the binding of the target sequences to the microsphere This produces a higher concentration of dye surrounding the microsphere than in the solution generally
  • a few demonstrated probe and target sequences see Ferguson, J A et al Nature Biotechnology, Vol 14, Dec 1996, are listed below in Table V
  • Hybridization indicators preferentially associate with double stranded nucleic acid
  • Hybridization indicators include mtercalators and minor and/or major groove binding moieties
  • mtercalators may be used, since intercalation generally only occurs in the presence of double stranded nucleic acid, only in the presence of target hybridization will the label light up
  • sensors may be made to detect nucleic acids, proteins (including enzyme sensors and immunosensors), lipids, carbohydrates, etc, similarly, these sensors may include bioactive agents that are nucleic acids, proteins, lipids, carbohydrates, etc
  • a single array sensor may contain different binding ligands for multiple types of analytes, for example, an array sensor for HIV may contain multiple nucleic acid probes for direct detection of the viral genome, protein binding ligands for direct detection of the viral particle, immuno-components for the detection of anti-HIV antibodies, etc
  • compositions of the invention may include other components, such as light sources, optical components such as lenses and filters, detectors, computer components for data analysis, etc.
  • the arrays of the present invention are constructed such that information about the identity of the bioactive agent is built into the array, such that the random deposition of the beads on the surface of the substrate can be "decoded” to allow identification of the bioactive agent at all positions This may be done in a variety of ways
  • the beads are loaded onto the substrate and then the array is decoded, prior to running the assay This is done by detecting the optical signature associated with the bead at each site on the array This may be done all at once, if unique optical signatures are used, or sequentially, as is generally outlined above for the "reuse" of sets of optical signatures Alternatively, decoding may occur after the assay is run
  • compositions find use in a number of applications
  • a sample containing a target analyte (whether for detection of the target analyte or screening for binding partners of the target analyte) is added to the array, under conditions suitable for binding of the target analyte to at least one of the bioactive agents, i e generally physiological conditions
  • the presence or absence of the target analyte is then detected
  • this may be done in a variety of ways, generally through the use of a change in an optical signal
  • This change can occur via many different mechanisms
  • a few examples include the binding of a dye-tagged analyte to the bead, the production of a dye species on or near the beads, the destruction of an existing dye species, a change in the optical signature upon analyte interaction with dye on bead, or any other optical interrogatable event
  • the change in optical signal occurs as a result of the binding of a target analyte that is labeled, either directly or indirectly, with a detectable label, preferably an optical label such as a fluorochrome
  • a detectable label preferably an optical label such as a fluorochrome
  • a protemaceous target analyte when used, it may be either directly labeled with a fluor, or indirectly, for example through the use of a labeled antibody
  • nucleic acids are easily labeled with fluorochromes, for example during PCR amplification as is known in the art
  • an intercalating dye e , ethidium bromide
  • the target analyte such as an enzyme generates a species (for example, a fluorescent product) that is either directly or indirectly detectable optically
  • a change in the optical signature may be the basis of the optical signal
  • the interaction of some chemical target analytes with some fluorescent dyes on the beads may alter the optical signature, thus generating a different optical signal
  • fluorophore denvatized receptors may be used in which the binding of the ligand alters the signal
  • sensor redundancy is used
  • a plurality of sensor elements, e g beads, comprising identical bioactive agents are used That is, each subpopulation comprises a plurality of beads comprising identical bioactive agents (e g binding ligands)
  • each subpopulation comprises a plurality of beads comprising identical bioactive agents (e g binding ligands)
  • bioactive agents e g binding ligands
  • a plurality of identical sensor elements are used as will be appreciated by those in the art, the number of identical sensor elements will vary with the application and use of the sensor array In general, anywhere from 2 to thousands may be used, with from 2 to 100 being preferred, 2 to 50 being particularly preferred and from 5 to 20 being especially preferred In general, preliminary results indicate that roughly 10 beads gives a sufficient advantage, although for some applications, more identical sensor elements can be used
  • the optical response signals from a plurality of sensor beads within each bead subpopulation can be manipulated and analyzed in a wide variety of ways, including baseline adjustment, averaging, standard deviation analysis, distribution and cluster analysis, confidence interval analysis, mean testing, etc
  • the first manipulation of the optical response signals is an optional baseline adjustment
  • the standardized optical responses are adjusted to start at a value of 0 0 by subtracting the integer 1 0 from all data points Doing this allows the baseline-loop data to remain at zero even when summed together and the random response signal noise is canceled out
  • the vapor pulse-loop temporal region frequently exhibits a characteristic change in response, either positive, negative or neutral, prior to the vapor pulse and often requires a baseline adjustment to overcome noise associated with drift in the first few data points due to charge buildup in the CCD camera If no drift is present, typically the baseline from the first data point for each bead sensor is subtracted from all the response data for the same bead If drift is observed, the average baseline from the first ten data points for each bead sensor is substracted from the all the response data for the same bead By applying this baseline adjustment, when multiple bead responses are added together they can be amplified while the baseline remains
  • signal summing is done by simply adding the intensity values of all responses at each time point, generating a new temporal response comprised of the sum of all bead responses These values can be baseline-adjusted or raw As for all the analyses described herein, signal summing can be performed in real time or during post-data acquisition data reduction and analysis In one embodiment, signal summing is performed with a commercial spreadsheet program (Excel, Microsoft, Redmond, WA) after optical response data is collected
  • cummulative response data is generated by simply adding all data points in successive time intervals This final column, comprised of the sum of all data points at a particular time interval, may then be compared or plotted with the individual bead responses to determine the extent of signal enhancement or improved signal-to-noise ratios as shown in Figs 14 and 15
  • the mean of the subpopulation (i e the plurality of identical beads) is determined, using the well known Equation 1
  • the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below)
  • the standard deviation of the subpopulation can be determined, generally using Equation 2 (for the entire subpopulation) and Equation 3 (for less than the entire subpopulation) Equation 2
  • the subpopulation may be redefined to exclude some beads if necessary (for example for obvious outliers, as discussed below)
  • statistical analyses are done to evaluate whether a particular data point has statistical validty within a subpopulation by using techniques including, but not limited to, t distribution and cluster analysis This may be done to statistically discard outliers that may otherwise skew the result and increase the signal-to-noise ratio of any particular experiment This may be done using Equation 4 Equation 4
  • the quality of the data is evaluated using confidence intervals, as is known in the art Confidence intervals can be used to facilitate more comprehensive data processing to measure the statistical validity of a result
  • statistical parameters of a subpopulation of beads are used to do hypothesis testing
  • tests concerning means also called mean testing
  • statistical evaluation is done to determine whether two subpopulations are different For example, one sample could be compared with another sample for each subpopulation within an array to determine if the variation is statistically significant
  • mean testing can also be used to differentiate two different assays that share the same code If the two assays give results that are statistically distinct from each other, then the subpopulations that share a common code can be distinguished from each other on the basis of the assay and the mean test, shown below in Equation 5
  • analyzing the distribution of individual members of a subpopulation of sensor elements may be done For example, a subpopulation distribution can be evaluated to determine whether the distribution is binomial, Poisson, hypergeometnc, etc
  • a preferred embodiment utilizes a plurality of sensor elements that are directed to a single target analyte but yet are not identical
  • a single target nucleic acid analyte may have two or more sensor elements each comprising a different probe This adds a level of confidence as non-specific binding interactions can be statistically minimized
  • the redundant nucleic acid probes may be overlapping, adjacent, or spatially separated However, it is preferred that two probes do not compete for a single binding site, so adjacent or separated probes are preferred
  • preferred embodiments utilize bioactive agent binding agents that bind to different parts of the target For example, when antibodies (or antibody fragments) are used as bioactive agents for the binding of target proteins, preferred embodiments utilize antibodies to different epitopes
  • a plurality of different sensor elements may be used, with from about 2 to about 20 being preferred, and from about 2 to about 10 being especially preferred, and from 2 to about 5 being particularly preferred, including 2, 3, 4 or 5 However, as above, more may also be used, depending on the application
  • Bead summing when beads are used, is the increase in sensitivity that can occur Detection limits in the zeptomole range can be observed
  • the presence or absence of the target analyte may be done using changes in other optical or non-optical signals, including, but not limited to, surface enhanced Raman spectroscopy, surface plasmon resonance, radioactivity, etc
  • the assays may be run under a variety of experimental conditions, as will be appreciated by those in the art
  • a variety of other reagents may be included in the screening assays
  • These include reagents like salts, neutral proteins, e g albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions
  • reagents that otherwise improve the efficiency of the assay such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc , may be used
  • the mixture of components may be added in any order that provides for the requisite binding
  • Various blocking and washing steps may be utilized as is known in the art
  • the compositions are used to probe a sample solution for the presence or absence of a target analyte
  • target analyte or “analyte” or grammatical equivalents herein is meant any atom, molecule, ion, molecular ion, compound or particle to be either detected or evaluated for binding partners
  • a large number of analytes may be used in the present invention, basically, any target analyte can be used which binds a bioactive agent or for which a binding partner (i e drug candidate) is sought
  • Suitable analytes include organic and inorganic molecules, including biomolecules
  • suitable target analytes include, but are not limited to, an environmental pollutant (including pesticides, insecticides, toxins, etc ), a chemical (including solvents, polymers, organic materials, etc ), therapeutic molecules (including therapeutic and abused drugs, antibiotics, etc ), biomolecule
  • the target analyte is a protein
  • Suitable protein target analytes include, but are not limited to, (1 ) immunoglobulms, (2) enzymes (and other proteins), (3) hormones and cytokmes (many of which serve as ligands for cellular receptors), and (4) other proteins
  • the target analyte is a nucleic acid
  • the probes are used in genetic diagnosis
  • probes can be made using the techniques disclosed herein to detect target sequences such as the gene for nonpolyposis colon cancer, the BRCA1 breast cancer gene, P53, which is a gene associated with a variety of cancers, the Apo E4 gene that indicates a greater risk of Alzheimer's disease, allowing for easy presymptomatic screening of patients, mutations in the cystic fibrosis gene, or any of the others well known in the art
  • viral and bacterial detection is done using the complexes of the invention
  • probes are designed to detect target sequences from a variety of bacteria and viruses
  • current blood-screening techniques rely on the detection of anti-
  • HIV antibodies The methods disclosed herein allow for direct screening of clinical samples to detect HIV nucleic acid sequences, particularly highly conserved HIV sequences In addition, this allows direct monitoring of circulating virus within a patient as an improved method of assessing the efficacy of anti-viral therapies Similarly, viruses associated with leukemia, HTLV-I and HTLV-II, may be detected in this way Bacterial infections such as tuberculosis, clymidia and other sexually transmitted diseases, may also be detected.
  • the nucleic acids of the invention find use as probes for toxic bacteria in the screening of water and food samples For example, samples may be treated to lyse the bacteria to release its nucleic acid, and then probes designed to recognize bacterial strains, including, but not limited to, such pathogenic strains as, Salmonella, Campylobader, Vibrio cholerae, Leishmama, enterotoxic strains of E coli, and Legionnaire's disease bacteria Similarly, bioremediation strategies may be evaluated using the compositions
  • the probes are used for forensic "DNA fingerprinting" to match crime-scene DNA against samples taken from victims and suspects
  • the probes in an array are used for sequencing by hybridization
  • the present invention also finds use as a methodology for the detection of mutations or mismatches in target nucleic acid sequences
  • STRs short tandem repeats
  • SNPs single nucleotide polymorphisms
  • compositions of the invention are used to screen bioactive agents to find an agent that will bind, and preferably modify the function of, a target molecule
  • an agent that will bind, and preferably modify the function of, a target molecule As above, a wide variety of different assay formats may be run, as will be appreciated by those in the art Generally, the target analyte for which a binding partner is desired is labeled, binding of the target analyte by the bioactive agent results in the recruitment of the label to the bead, with subsequent detection
  • the binding of the bioactive agent and the target analyte is specific, that is, the bioactive agent specifically binds to the target analyte
  • specifically bind herein is meant that the agent binds the analyte, with specificity sufficient to differentiate between the analyte and other components or contaminants of the test sample
  • binding which is not highly specific, for example, the systems may use different binding ligands, for example an array of different ligands, and detection of any particular analyte is via its "signature" of binding to a panel of binding ligands, similar to the manner in which "electronic noses" work This finds particular utility in the detection of chemical analytes
  • the binding should be sufficient to remain bound under the conditions of the assay, including wash steps to remove non-specific binding, although in some embodiments, wash steps are not desired, i e for detecting low affinity binding partners
  • wash steps are not desired, i e for detecting low affinity binding partners
  • a range of ratios of light intensities are selected that are representative of the optical signature for the dye ratio of the subpopulation based on the quantum yield of the two dyes
  • Bioactive agent B-Galactosidase, Target substrate fluorescein di-B-galactopyranoside (FDG)
  • Target substrate fluorescein di-B-D-glucuronide (FDGicu)
  • Reporter dye ratio 1 10 ratio of DilC TRC, which translates to an optical signature of DilC A mtensitv-ave DilC background 0 2136 + 0 03
  • the respective enzymes on the microspheres catalyze the breakdown of the substrates producing fluorescein which is fluorescent, emitting light at 530 nanometers when excited at 490 nm
  • the production of fluorescein localized to particular beads is then monitored
  • the localization of fluorescein around the microspheres is increased by using a substrate solution of 90% glycerol and 10% substrate The glycerol inhibits the generated fluorescein from diffusing away from the microsphere reaction sites
  • the substrate solution is then introduced and CCD images acquired every 30 seconds to a minute for 30 minutes While illuminating the microspheres with 490 nm light and collecting emissions in the 530 nm range Fiber is then placed back in the buffer solution and another background image captured Those beads that generate a signal indicative of fluorescein production are decoded Depending in the ratio of the intensity of light from the two reporter dyes, DilC TRC, the bioactive agent of the optically active beads may be decoded according to the following table
  • Figs 8A-8C are images generated by the CCD 236 when the bead populations are exposed to fluorescein diphosphate
  • Fig 8A illustrates the signals from the alkaline phosphatase microspheres when excited at 490 nm and recording emissions at 530 nm, emissions at this wavelength being indicative of fluorescein production
  • Fig 8B shows the image captured by the CCD when the microspheres are excited at 577 nm and emissions at 670 nm are recorded This wavelength is an encoding wavelength indicative of the concentration of DilC on the microspheres
  • Fig 8C shows the image when the microspheres are excited with 577 nm light and emissions in the 610 nm range are recorded being indicative of the concentration of TRC in the microspheres
  • Figs 9A and 9B are images when the microspheres are exposed to fluorescein ⁇ -d- gatactosidase Fig 9A shows emissions at 530 nm indicative of the fluorescein production, and Fig 9B shows light emitted at the 670 nm range indicative of the presence of DilC
  • micrographs Fig 8A-8C and 9A-9B illustrate that fluorescein production around the microspheres may be detected as an optical signature change indicative of reactions involving the bioactive agent of the microspheres
  • the micrographs also illustrate that the optical signatures may be decoded to determine the chemical functionalities on each microsphere
  • subpopulation A Three separate subpopulations of beads were used In subpopulation A, xrabbit antibodies (Ab) were affixed to the surface of the microspheres, in subpopulation B, xgoat antibodies were affixed to the microspheres, and in subpopulation C, xmouse antibodies were affixed to the microspheres These three separate subpopulations were identified using a DilC TRC encoding ratio similar to that in the previously described experiment

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